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Abstract

Background—Calmodulin (CaM) mutations are associated with severe forms of long QT syndrome and catecholaminergic polymorphic ventricular tachycardia (CPVT). CaM mutations are found in 13% of genotype-negative long QT syndrome patients, but the prevalence of CaM mutations in genotype-negative CPVT patients is unknown. Here, we identify and characterize CaM mutations in 12 patients with genotype-negative but clinically diagnosed CPVT.

Conclusions—We discovered a novel CPVT mutation in the CALM3 gene that shares functional characteristics with established CPVT-associated mutations in CALM1. A small proportion of A103V-CaM is sufficient to evoke arrhythmogenic Ca disturbances via ryanodine receptor 2 dysregulation, which explains the autosomal dominant inheritance.

WHAT IS KNOWN

Calmodulin mutations have been associated with severe forms of long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT).

In previous reports, calmodulin mutations were found in 13% of genotype-negative LQTS patients.

WHAT THE STUDY ADDS

This report identifies and characterizes a novel calmodulin mutation in the CALM3 gene (A103V-CaM) in 1 out of 12 patients with genotype-negative but clinically-diagnosed CPVT.

A103V-CaM shares in vitro functional characteristics with the two published and established CPVT-associated mutations, suggesting that it should be considered a CPVT-susceptibility mutation.

In vitro functional studies of spontaneous Ca2+ release events in combination with action potential measurements can be used to rapidly assess the molecular mechanism underlying the pathogenicity of calmodulin variants.

Calmodulin (CaM) is an essential Ca-binding protein with multiple cellular targets. CaM has 4 EF-hand Ca-binding motifs located in 2 N-terminal (CaM-N) and 2 C-terminal (CaM-C) globular domains, which are connected by a flexible linker.1 At high [Ca], Ca binds cooperatively to CaM, inducing a conformational change that transduces Ca signals in a wide range of biological processes, including inflammation, muscle contraction, memory, metabolism, and immune responses. Recently, CaM missense mutations have been identified in patients with severe ventricular arrhythmia and sudden death susceptibility.2–4 Humans have 3 CaM genes (CALM1, CALM2, and CALM3) encoding for a perfectly conserved sequence of amino acids, and all 3 CaM genes are expressed in the heart.5

CaM regulates a variety of ion channels in cardiac myocytes. Among them, changes in CaM regulation of the L-type calcium channel (LTCC) and ryanodine receptor 2 (RyR2) have been associated with autosomal dominant syndrome of sudden death that can present with clinical features of long QT syndrome (LQTS) or catecholaminergic-induced polymorphic ventricular tachycardia (CPVT).2–4 CaM is the main Ca sensor for the LTCC,6,7 and mutations that lead to a reduction in CaM Ca-binding affinity impair Ca-dependent inactivation of LTCCs and cause action potential (AP) prolongation, which can explain the LQTS phenotype.8 CaM also binds to RyR2 stoichiometrically (4 CaMs per tetrameric RyR2)9 and inhibits RyR2 opening at all [Ca], which regulates sarcoplasmic reticulum (SR) Ca release.10 Mutations that cause defective CaM-mediated RyR2 inactivation may contribute to RyR2 leakiness, promoting significantly higher spontaneous Ca wave and spark activity, a typical cellular phenotype of CPVT.

Here, we tested the prevalence of CaM mutations in a cohort of 12 patients with genotype-negative CPVT. We identified a novel CaM mutation—A103V—in CALM3, in one of the 12 patients. We then characterized the functional effects of the novel A103V mutant CaM on Ca-binding affinity, RyR2 binding, Ca handling, and the LTCC and compared the effects of A103V to those of the only 2 other CaM mutants associated with CPVT (N54I and N98S). Our data suggest that the CALM3-A103V mutation underlies the patient’s CPVT.

Methods

Study Subjects

The study population consisted of 12 unrelated patients with genotype-negative but clinically diagnosed CPVT who were referred to the Windland Smith Rice Sudden Death Genomics Laboratory at Mayo Clinic, Rochester, Minnesota, for genetic testing with robust evidence of CPVT with either an abnormal stress test or documented ventricular ectopy (Table). The 12 patients were found to be genotype-negative after CPVT mutational analysis of the 2 major CPVT genes, RYR2 and CASQ2. This study was approved by the Mayo Foundation Institutional Review Board, and informed consent was obtained for all patients.

Animal Use

The use of animals in this study was approved by the Animal Care and Use Committees of Vanderbilt University, Nashville, TN, and the University of Minnesota, MN, and performed in accordance with National Institutes of Health guidelines.

Measurement of Ca Binding to CaM

To study the Ca binding affinity of the mutant A103V-CaM, we bacterially expressed and purified recombinant wild-type (WT) and mutant CaMs (N54I, N98S, D96V, and A103V), as reported previously.3 To summarize, the recombinant CaM cDNA subcloned into a pET15b vector was mutated using QuikChange site directed mutagenesis. WT and mutant CaMs were expressed in Escherichia coli BL21 (DE3) cells and purified by hydrophobic chromatography using a phenyl sepharose column. To minimize Ca, the proteins were dialyzed 4× at 4°C twice in 50 mmol/L HEPES at pH 7.4, 100 mmol/L KCl, and 5 mmol/L EGTA and twice more with the same buffer, except EGTA was lowered to 0.05 mmol/L. The molecular mass of all proteins was confirmed using negative electro-spray mass spectroscopy. Ca-binding affinities for WT and A103V-CaM proteins were determined as described.3 Briefly, macroscopic-binding constants for the pairs of Ca-binding sites in CaM-N and CaM-C were measured by monitoring the intrinsic tyrosine and phenylalanine fluorescence of the protein during the course of a Ca titration. Free [Ca] was determined using the fluorescent dye fluo-5N. Data were analyzed by plotting the normalized fluorescence signal versus free [Ca] and fitting to the model-independent, 2-site Adair function. The dissociation constants (Kd) for each domain are reported as the average value for the pair of sites by taking the square root of K2 from the Adair equation.

Ventricular Myocytes Isolation

Single ventricular myocytes from 10- to 16-week-old C57BL/6 mice were isolated by enzymatic digestion using collagenase as previously described.12 Briefly, the ascending aorta was cannulated and the heart was perfused with collagenase (Worthington type II) and protease at 36 to 37°C. After perfusion, the hearts were cut up, chopped into small pieces, filtered through a 250 μm mesh, and washed twice in a standard Tyrode solution containing 0.2 mmol/L of CaCl2 to disperse the isolated left ventricular myocytes. Finally, the cells were resuspended in a solution containing 0.6 mmol/L of CaCl2.

Ca Wave Measurement

For Ca wave measurements, cardiomyocytes were first exposed to a Ca-free relaxing solution and then permeabilized with saponin (40 μg/mL) for 60 seconds and placed in internal solution composed (in mmol/L) of 120 K-aspartate, 15 KCl, 5 KH2PO4, 0.75 MgCl2, 4% dextran, 10 HEPES, 5 Mg2ATP, 10 glutathione (reduced), 0.025 Fluo-4, and 10 phosphocreatine (di-Na). These solutions also contained 10 U/mL creatine phosphokinase and had free [Ca] =120 nmol/L. To allow equilibration of CaM binding to cellular targets, all Ca wave recordings were done after 30-minute incubation with either WT or CPVT mutant CaMs (A103V, N54I, or N98s). Free [CaM] was kept at the physiological concentration of 100 nM.13 Ca waves in myocytes were imaged with a confocal microscope (LSM 510 Zeiss) in line-scan mode. Ca wave analysis was performed as described.4 For the mixing studies, cells were incubated with WT-CaM at a final concentration of 100 nmol/L or with a mixture of 75% WT-CaM and 25% CPVT-CaM mutants A103V, N54I, or N98S (final concentration 100 nmol/L). Given the variability between different experimental days, the Ca wave frequency and amplitude data were normalized to the mean of WT group obtained on the same day.

Inactivation of LTCCs

Inactivation of L-type Ca currents (ICaL) was studied in freshly isolated murine ventricular myocytes using whole-cell patch clamp technique. CaM (final concentration 6 µmol/L) was added to the internal solution composed (in mmol/L) of 110 CsCl, 1 MgCl2, 5 MgATP, 0.2 cAMP, 14 EGTA, and 20 HEPES and then dialyzed into the cell via patch pipette using positive pressure. The external solution was composed (in mmol/L) of 134 NaCl, 5 CsCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES. Currents were elicited with 500-ms depolarizing pulses to 0 mV from holding potential H=−70 mV applied every 2 minutes to track the effect of CaM over time as it diffuses into the cell. Usually, the effect of CaM on ICaL inactivation was found to be reaching its maximum in 4 to 6 minutes after start of infusion. A 15-ms prepulse to −40 mV was applied before the test pulse to inactivate Na currents. Cells were pretreated with ryanodine (50 µmol/L) and thapsigargin (10 µmol/L) for 30 minutes before every experiment to prevent SR Ca release. All experiments were conducted at room temperature.

AP Measurements

For these experiments, cells were studied in current-clamp mode. Ventricular APs were measured using pipette solutions containing (in mmol/L) potassium gluconate, 110; NaCl, 5; KCl, 10; EGTA, 0.5; HEPES, 10; MgATP, 5; and cAMP, 0.2 pH adjusted to 7.2 with KOH. 6 µmol/L of WT, A103V mutant or D96V mutant CaM was added to the pipette solution and dialyzed into the cell via patch pipette using positive pressure. Whole-cell patches were established in control Tyrode solution containing (mmol/L) NaCl, 134; KCl, 5.4; MgCl2, 1; CaCl2, 2; HEPES, 10; and glucose, 10 and pH adjusted to 7.4 with NaOH. A train of 4 APs (1 Hz) was triggered by application of a 2-ms current injection 20% above threshold (usually 0.3–0.4 nA). Resting potential and AP duration (APD) measured at 50% and 90% repolarization (APD50 and APD90, respectively) were measured from the last paced AP for every cell. The incidence of early afterdepolarizations was quantified for each cell during the pacing train. The incidence of delayed afterdepolarizations (DADs) and spontaneous beats triggered by DADs was calculated during the 45-second period after the pacing train.

Statistical Analysis

Data are presented as means±SD. Gaussian distribution of the samples was assessed by D’Agostino-Pearson test. Then, statistical significance was evaluated by 1-way analysis of variance followed by Tukey post test in those samples that followed a normal distribution or by Kruskal–Wallis followed by Dunn’s post test. For statistical comparison of early afterdepolarization activity occurrence, the Fisher’s exact test was used, and the data are presented as percentage. A value of P<0.05 was considered statistically significant.

Results

Clinical Studies

Our genotype-negative CPVT cohort consisted of 12 individuals, of which 25% were males, and the average age at diagnosis was 20±12 years. Overall, 83% experienced syncope, 17% cardiac arrest, and 25% had a positive family history of cardiac arrhythmias or sudden unexplained death (Table). Denaturing high-performance liquid chromatography revealed one novel CaM missense mutation, p.A103V in CALM3, in one patient.

The A103V-CALM3-positive subject is currently a 31-year-old female who presented at 10 years of age with a loss of consciousness during exertion. Subsequently, between the ages of 10 and 14, she experienced a dozen additional syncopal episodes, many of which took place during exertion. Two of the syncopal events resulted in loss of bladder function and one required cardiopulmonary resuscitation. Because of these episodes, she had an ECG taken, which was unremarkable except for a prominent U wave. Her initial stress test in 1994 recorded single premature ventricular contractions (PVCs) in isolation during exercise. The ectopy increased to PVCs in bigeminy. During epinephrine infusion, she also had bigeminal PVCs and a brief run of nonsustained ventricular tachycardia triplets. In 1994, she was diagnosed originally with atypical LQTS and treated with beta blockers. Treadmill stress tests while on beta blockers still evidenced PVCs in bigeminy and couplets but with lower frequency (Figure 1). For the past 20 years, she has been event free while being fully compliant on beta blockers. In 2000, CPVT instead of atypical LQTS was considered as the likely diagnosis, but the patient tested negative for the known CPVT-susceptibility genes.

Stress test for patient with A103V-CaM. A representative stress test for the patient harboring the A103V-CaM variant while on beta blocker therapy. Shown are the rhythm strips for the baseline, stage 2 with the onset of premature ventricular contractions (PVCs), a couplet, the peak exercise heart rate, and 3 minutes into recovery. A103V-CaM indicates calmodulin mutation in the CALM3 gene; and CaM, calmodulin.

There is no family history of arrhythmias or sudden deaths. However, her mother described several fainting episodes when she was school age. Her mother and father underwent stress testing, and although her father’s test was normal, her mother’s test revealed exercise-induced PVCs. Mutation-specific testing established that her mother was also heterozygous for A103V-CALM3 while her father was negative.

Effect of A103V-CaM on Ca Binding

CaM binds Ca at 2 higher-affinity Ca-binding sites situated in the C-domain (EF-hand III and EF-hand IV) and 2 lower-affinity sites in the N-domain (EF-hand I and EF-hand II).14 As expected based on the site of the mutation in EF-hand III, this novel CALM3 A103V mutant has reduced Ca-binding affinity in the C-domain (3-fold reduction), whereas the mutation did not significantly alter Ca binding to the N-domain (Figure 2). This effect in Ca-binding affinity was different from that found with N54I, an established CPVT-related CaM mutant located in the EF-hand II that had no effect on Ca-binding affinity in either domain. However, the modestly reduced Ca-binding affinity was similar to that of the only other known CPVT-linked CaM mutant that is also located in EF-hand III (N98S).4

Ca titration curves for wild-type (WT) catecholaminergic polymorphic ventricular tachycardia (CPVT) calmodulin (CaM) mutant (A103V) for the CaM-C and CaM-N domain. Dissociation constants (Kd, in μM) were derived for each domain by following intrinsic Tyr and Phe fluorescence for the N-domain and the C-domain, respectively, and fitting to a standard binding equation. The A103V-CaM mutant reduced Ca binding affinity of CaM C-domain (3-fold reduction compared with WT-CaM), whereas N-domain Ca binding was unchanged. Values are averages of 3 experiments, and error was determined by analysis of the curve fits. A103V-CaM indicates calmodulin mutation in the CALM3 gene; and CaM, calmodulin.

A103V-CaM Promotes Ca Sparks’ Activity and Reduces SR Ca Load

Our previous studies have shown that the 2 known CPVT-linked CaM mutants N54I and N98S both cause RyR2 activation.4 Hence, we next measured the effect of A103V-CaM on Ca sparks—a measure of RyR2 Ca release channel activity in cardiomyocytes15—and compared the results with the CPVT-linked N54I-CaM and N98S-CaM (Figure 3). To avoid wave propagation, free [Ca] was kept at 50 nmol/L with strong buffering by 0.5 mmol/L EGTA. The cardiomyocytes were permeabilized with saponin to prevent any influence of membrane ion channels. Compared with WT-CaM, A103V-CaM increased spark frequency (Figure 3B) to a similar level as N54I-CaM or N98S-CaM without modifying any other spark parameter, such as amplitude, duration, or width (Figure 3C–3E). As a result of increased SR Ca leak, all 3 CaM mutants reduced SR Ca content assessed by rapid caffeine application (Figure 3F–3G).

A103V-CaM Promotes Ca Waves

We next examined the consequences of CPVT-CaMs on arrhythmogenic Ca waves. Experiments were performed at physiological free [CaM] of 100 nmol/L and diastolic free [Ca] of 120 nmol/L. Under those conditions, cardiomyocytes incubated with A103V-CaM exhibited increased spontaneous Ca release in the form of regular propagated Ca waves compared with WT-CaM (Figure 4A and 4C). The effect of A103V-CaM was similar to the effect obtained on incubation with N54I or N98S. A103V-CaM but not N54I or N98S-CaM also increased the amplitude of Ca waves (Figure 4).

A103V-CaM Exhibits Dominant-Activating Effects on Ca Waves

As only 1 out of 6 CaM alleles of our proband hosts the A103V-CaM missense mutation, we next tested whether a small fraction of mutant CaM in the presence of WT-CaM (25% mutant, 75% WT, final concentration 100 nmol/L) can exert a dominant effect on RyR2 function. We found that in the presence of 3-fold excess of WT-CaM (75%), A103V-CaM promoted significantly higher Ca wave frequencies4 compared with WT-CaM. The dominant effect of A103V-CaM was as strong as that of N54I-CaM and N98S-CaM, the only 2 other CaM mutants implicated in CPVT.

RyR2 Binding

We next used a FRET-based assay to specifically detect CaM/RyR2 binding in SR vesicles.11,16 RyR2-specific CaM binding was measured by FRET between D-FKBP and A-CaM. Figure 5 illustrates how A103V- and WT-CaM compete with 100 nmol/L A-CaM and reduce FRET at either 30 nmol/L or 30 μmol/L [Ca]. These results indicate that CaM-A103V binds to RyR2 with an affinity that is comparable to WT-CaM, both at nM and μM [Ca] relevant to diastolic and systolic conditions in the heart.

The A103V mutation does not significantly change the affinity of CaM for RyR2. Both CaM species were equally effective in displacing acceptor-labeled CaM (A-CaM) at nM and μM [Ca] relevant to diastolic and systolic conditions in the heart. CaM indicates calmodulin.

A103V-CaM Modestly Disrupts Inactivation of LTCCs

Alterations in Ca binding to CaM may disrupt Ca-dependent inactivation of LTCC in the heart.6,17 Thus, we next tested the effect of A103V in ventricular myocytes dialyzed with WT or mutant CaMs. A103V showed a trend to slightly impaired LTCC inactivation without affecting peak currents compared with WT in a similar way as N98S-CaM, whereas N54I had no effect on LTCC inactivation (Figure 6). However, the effect of A103V-CaM on LTCC inactivation was much smaller than that of D96V-CaM, a mutation associated with severe LQTS but not CPVT in humans.

A103V-CaM showed a trend to disrupt L-type Ca channels (LTCC) inactivation. A, Representative examples of traces for each experimental group. B, Average current densities (pA/pF) obtained in cells dialyzed with either WT-CaM, A103V-CaM, N54I-CaM, N98S-CaM (catecholaminergic polymorphic ventricular tachycardia [CPVT] mutants) or D96V-CaM (LQTS mutant). CaM mutants had no effect on the peak current density. C, Effect of A103V-CaM, N54I-CaM, N98S, and D96V-CaM on inactivation time constant of the LTCC compared with WT-CaM. A103V-CaM and N98S-CaM showed a trend to impair LTCC inactivation, whereas N54I-CaM did not affect it at all. However, compared with an LQTS-associated CaM mutation (D96V-CaM), A103V-CaM had a smaller effect on LTCC inactivation. Data are mean±SD (WT-CaM, A103V-CaM, and N54I-CaM, n=6; N98S-CaM and D96V-CaM, n=4. *P<0.05 vs WT-CaM; ††P<0.01 vs N54I-CaM). A103V-CaM indicates calmodulin mutation in the CALM3 gene; CaM, calmodulin; and LQTS, long QT syndrome.

A103V-CaM Does Not Modify APD but Promotes DAD and Triggered Beats

We next evaluated the effect of the mutant CaMs on the APD in intact cardiomyocytes dialyzed with WT, A103V-CaM, or the LQTS-associated D96V-CaM. A103V-CaM did not have a significant effect on APD50 or APD90 compared with WT, whereas D96V dramatically increased both APD50 and APD90 (Figure 7A). Consistent with its clinical phenotype of LQTS, early afterdepolarizations were observed in 36% of cells dialyzed with D96V-CaM. Early afterdepolarizations were not found in cells dialyzed with WT or A103V-CaM (Figure 7B). Finally, we quantified the incidence of DADs and triggered beats after the 1 Hz pacing train. Consistent with the activation of spontaneous Ca release observed in permeabilized myocytes (Figures 3 and 4), only the A103V-CaM mutant associated with CPVT caused spontaneous beats triggered by DADs (Figure 7C).

A103V-CaM does not modify action potential duration (APD) but induces spontaneous beats triggered by delayed afterdepolarizations (DADs). A, Top, Representative examples of action potential (AP) records for each experimental group. Bottom, Average APD measured at 50% (APD50, left) and 90% (APD90, right) of repolarization. B, Top, Representative example of prolonged APs with early afterdepolarizations (EADs) recorded from a cardiomyocyte dialyzed with D96V-CaM. Bottom, Percentage of cardiomyocytes exhibiting EADs during the pacing train. C, Top, Representative examples of DADs and triggered beats recorded from a cardiomyocyte dialyzed with A103V-CaM. Bottom, Averaged number of DADs (left) and triggered beats (right) during the first 45 s after the pacing train in cardiomyocytes dialyzed with either wild-type (WT) or mutant CaMs. Data are mean±SD. (WT-CaM n=7, A103V-CaM n=14, and D96V-CaM n=12; *P<0.05, **P<0.01 vs WT-CaM; †P<0.05 vs A103V-CaM). A103V-CaM indicates calmodulin mutation in the CALM3 gene; and CaM, calmodulin.

Discussion

The major finding reported here is the discovery of a novel CPVT mutation—CaM-A103V—in the CALM3 gene: a gene that up to now had not been linked to CPVT. Furthermore, finding CaM mutations in 1 out of 12 patients (8%) with major CPVT genotype-negative but clinically diagnosed CPVT suggests that the prevalence of CaM mutations in CPVT may be higher than previously thought. Because CaM mutations have also been identified in 13% of genotype-negative LQTS patients,18 and in a family with idiopathic ventricular fibrillation,19 CaM mutations should be considered a significant contributor to genetic sudden death syndromes.

The cellular mechanisms involved in the arrhythmia susceptibility in the setting of CaM mutations are complex because of the multiple targets of this protein in the cell. For example, dysfunctional CaM mutants can impair Ca-dependent inactivation of LTCCs, leading to increased depolarizing current during the plateau phase of the cardiac AP.3 CaM mutations can also lead to aberrant regulation of the RyR2 Ca release.4,9 Our in vitro investigation demonstrated that CaM-A103V has an activating effect on RyR2 Ca release that is similar to that reported for 2 established CPVT-linked CaM mutations (N54I and N98S)4 and matches also the defect in Ca-handling observed in animal models of CPVT–linked mutations residing directly within either the cardiac RyR2 or calsequestrin.12,20 Given that models of human CPVT-linked mutations present higher rates of spontaneous Ca release and DADs21 and that drugs that suppress spontaneous SR Ca release in single cells are effective in preventing CPVT in animal models and patients,22 it is generally accepted that spontaneous Ca release from the SR is the underlying pathophysiological mechanism responsible for CPVT. In this regard, previous studies using CALM1 mutants (N54I and N98S) showed that both CPVT-CaMs bound with greater affinity to RyR2 than WT-CaM, but at the same time both failed to inhibit or directly activated RyR2, rendering them hyperactive and generating arrhythmogenic Ca waves.4

The novel CALM3 mutation A103V studied here also activates RyR2 Ca release channels, generating Ca waves and depleting SR Ca store. In fact, spontaneous Ca releases induced by this mutation generated DADs that triggered spontaneous APs (Figure 7C), which is consistent with the clinical CPVT phenotype. Similar to the previously reported CALM1 mutations, A103V-CaM exhibited dominant-activating effects on Ca waves when mixed with WT-CaM, which is consistent with an autosomal dominant inheritance pattern in humans.4 However, in contrast to the previously published CPVT CaM mutants, A103V did not exhibit increased RyR2 affinity. The significance of the latter finding is unclear, but could mean that only 1 mutant CaM out of 4 CaMs bound to the RyR2 complex is needed and sufficient to cause aberrant RyR2 regulation. The unchanged RyR2-binding affinity of A103V-CaM, compared with the increased affinity observed for the 2 other CPVT-linked CaM mutants N54I and N98S, might explain the milder phenotype of our 2 patients compared with patients carrying the previously reported CPVT-linked CALM1 mutations, especially because CALM3 mRNA transcript levels are reportedly higher than CALM1 and 2 mRNA transcripts levels in human hearts.3

Another important CaM target related to arrhythmogenesis is the LTCC (Cav1.2). Preassociation of Ca-free CaM with Cav1.2 regulates Ca-dependent channel inactivation.23 Thus, CaM mutations that show reduced Ca-binding affinity can disrupt Ca-dependent inactivation of LTCC. Although A103V-CaM mutant showed a trend to impair LTCC inactivation because of a small reduction in Ca-binding affinity, the effect is small compared with the published LQTS-associated CaM mutations (D96V, D130G F142L, and E141G).8,18 Furthermore, ICaL measurements were done with blocked SR, and unlike LQTS-associated CaM mutations, A103V enhances SR Ca release and, consequently, the Ca-dependent inactivation of LTCCs. In fact, we observed that the net effect of A103V-CaM on AP duration was minimal in contrast to the massive AP prolongation observed in cells dialyzed with the LQTS-linked D96V-CaM (Figure 7A),which is consistent with the lack of QT prolongation in the A103V mutation-positive patient. Nevertheless, the subtle impairment of Ca-binding affinity and LTCC inactivation (Figures 2 and 6) and the trend toward longer APD (Figure 7) caused by A103V-CaM may explain the U waves observed in the ECG of our patient. Notably, the CPVT-linked N98S-CaM mutation also causes some impairment of Ca binding4 and, hence, LTCC inactivation, with U waves reported in the first index case,2 and a full LQTS phenotype reported in a second individual who carried the same N98S mutation in CALM2.24

Study Limitations

CaM also binds other targets in the heart, and thus, we cannot exclude that other mechanisms contribute to the autosomal dominant inheritance of A103V-CaM mutation. However, its dominant activating effect on RyR2-mediated Ca release is likely sufficient to explain the CPVT phenotype of humans positive for the A103V-CaM mutation.

Conclusions

We find that the in vitro phenotype produced by A103V-CaM is analogous to the 2 published and established CPVT-linked CaM mutations, which suggests that A103V should be considered a CPVT-susceptibility mutation. Our results further indicate that in vitro functional studies of Ca release in combination with AP measurements in intact cardiomyocytes can be used to rapidly assess the molecular mechanism underlying the pathogenicity of CaM variants that will be discovered in the future. Just as CALM3 has been implicated recently in the pathogenesis of LQTS, thereby, completing the CaM-mediated LQTS trilogy,25,26 now the CaM-mediated CPVT trilogy has been completed with the discovery of A103V-CALM3.

Sources of Funding

This work was partly supported by the United States National Institutes of Health (HL88635, HL71670, and HL108173 to B.C. Knollmann); 5 F32 HL117612-02 to C.N. Johnson; T32 HL069764 (to F.R. Nitu); HL092097 and AG26160 to R.L. Cornea; by the American Heart Association (13IRG13680003 to B.C. Knollmann, 12POST12080080 to D.O. Kryshtal, and 15GRNT25610022 to R.L. Cornea); and by the Mayo Clinic Windland Smith Rice Comprehensive Sudden Cardiac Death Program to M.J. Ackerman.

Disclosures

M.J. Ackerman is a consultant for Boston Scientific, Gilead Sciences, Medtronic, and St Jude Medical. In addition, M.J. Ackerman and Mayo Clinic receive royalties from Transgenomics for their FAMILION-LQTS and FAMILION-CPVT genetic tests. However, none of these commercial entities had any role in this study. R.L. Cornea holds equity in and serves as an executive officer for Photonic Pharma LLC. This relationship has been reviewed and managed by the University of Minnesota. Photonic Pharma had no role in this study. The other authors report no conflicts.